MiR129-5p-loaded exosomes suppress seizure-associated neurodegeneration in status epilepticus model mice by inhibiting HMGB1/TLR4-mediated neuroinflammation

Background Neuroinflammation contributes to both epileptogenesis and the associated neurodegeneration, so regulation of inflammatory signaling is a potential strategy for suppressing epilepsy development and pathological progression. Exosomes are enriched in microRNAs (miRNAs), considered as vital communication tools between cells, which have been proven as potential therapeutic method for neurological disease. Here, we investigated the role of miR129-5p-loaded mesenchymal stem cell (MSC)-derived exosomes in status epilepticus (SE) mice model. Methods Mice were divided into four groups: untreated control (CON group), kainic acid (KA)-induced SE groups (KA group), control exosome injection (KA + Exo-con group), miR129-5p-loaded exosome injection (KA + Exo-miR129-5p group). Hippocampal expression levels of miR129-5p, HMGB1, and TLR4 were compared among groups. Nissl and Fluoro-jade B staining were conducted to evaluate neuronal damage. In addition, immunofluorescence staining for IBA-1 and GFAP was performed to assess glial cell activation, and inflammatory factor content was determined by ELISA. Hippocampal neurogenesis was assessed by BrdU staining. Results The expression of HMGB1 was increased after KA-induced SE and peaking at 48 h, while hippocampal miR129-5p expression decreased in SE mice. Exo-miR129-5p injection reversed KA-induced upregulation of hippocampal HMGB1 and TLR4, alleviated neuronal damage in the hippocampal CA3, reduced IBA-1 + and GFAP + staining intensity, suppressed SE-associated increases in inflammatory factors, and decreased BrdU + cell number in dentate gyrus. Conclusions Exosomes loaded with miR129-5p can protect neurons against SE-mediated degeneration by inhibiting the pro-inflammatory HMGB1/TLR4 signaling axis. Graphical Abstract


Introduction
Epilepsy is characterized by spontaneous and recurrent episodes of neuronal hyperactivity (seizures) that can lead to progressive neurological damage [1,2].Epileptic seizures are usually terminated by multiple endogenous homeostatic mechanisms, but these can be disrupted by chronic seizure activity, leading to potentially fatal SE, defined clinically by seizures lasting longer than 5 min or by more than one seizure within 5 min and leading to abnormally, prolonged seizures [3,4].The sustained seizure activity of SE can induce acute excitotoxic neuronal injury or death, neuroinflammation and secondary neuronal injury, and abnormal neurogenesis, ultimately leading to functional deficits and greater risk of future SE [3,5].Moreover, currently available antiepileptic drugs are only partially effective on reducing seizures and preventing SE, consequently, epilepsy remains a fatal neurological disorder [6].
Neuroinflammation is a major pathomechanism contributing to epilepsy progression and associated neurological dysfunction [7].Seizures induce an inflammatory cytokine storm characterized by activation of glial cells and release of pro-inflammatory factors such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α [8][9][10].These neuroinflammatory responses induce a progressive self-sustaining cycle of neuronal hyperexcitability, neuronal injury, and aberrant neural plasticity that ultimately reduces seizure threshold in susceptible regions such as the hippocampus [11].Accordingly, controlling inflammatory signaling may serve as an effective means to prevent epileptic seizures.Indeed, anti-inflammatory drugs have demonstrated anti-epileptic effects in animal models and human patients [12,13].Moreover, these agents can prevent neurodegeneration secondary to epilepsy-induced neuroinflammation [14].However, anti-inflammatory drugs have a myriad of deleterious side effects and suppress inflammatory signaling pathways for a relatively limited period of time.Thus, long-lasting strategies targeting epilepsy-associated neuroinflammatory pathways are required.
MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression primarily by binding to the 3′-UTR of complementary (target) mRNAs, thereby influencing a myriad of biological processes including cell proliferation and differentiation [15,16].Dysregulation of miRNAs is implicated in the pathogenesis of multiple central nervous system (CNS) diseases including epilepsy [17].Differential expression of numerous miRNAs has been detected in blood and brain samples of epileptic patients.However, free miRNAs are easily degraded in body fluids and by intracellular lysosomal pathways, limiting therapeutic applicability.Alternatively, it is possible to facilitate the transmission of miRNAs into target cells via exosomes, small (30-100 nm) vesicles containing proteins, lipids, and miRNAs secreted by most cell types [18].These structures can also pass through the blood-brain barrier (BBB), allowing delivery into the brain by systemic injection, while the double-layer lipid membrane both protects miRNAs from breakdown or chemical modification and promotes intracellular delivery via membrane fusion.The therapeutic potential of miRNA-loaded exosomes has been widely examined, including mesenchymal stem cell (MSC)-derived exosomes loaded with miR-133b in a rat model of intracerebral hemorrhage [19].
The miR-129 family member miR-129-5p is widely reported to suppress tumor formation [20] by negatively regulating the expression of high mobility group box 1 protein (HMGB1) [21], a multifunctional regulator of gene expression, including genes associated with neuroinflammation and oxidative stress.Moreover, HMGB1 is released by immune cells or glia cells and neurons in the CNS [22], and release is upregulated in the brain of epileptic patients and epileptic animal models [23][24][25], strongly implicating this protein in the pathophysiology of epilepsy.
Therefore, we can suggest a hypothesis that miR129-5p may decrease the release of HMGB1 and inflammatory factors in the epileptic brain, mitigating SE-induced neurological damage.We aim to explore the effect of miR129-5p-loaded exosomes on KA-induced SE and indicate the underlying mechanism.

MiR129-5p transfection of MSCs
Mesenchymal stromal cells (MSCs) were isolated and collected from 6 to 8-week-old male C57BL/6 J mice as described [26] and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin at 37 °C under a 5% CO 2 95% air atmosphere.Cultures were fed fresh DMEM 48 h after isolation and once every 2 days thereafter.At 90% confluence, the medium was replaced with DMEM plus exosome-free FBS and MSCs were transfected with miR126-5p mimic or mimic NC (Ruibo Biotechnology, Guangzhou, China) for 72 h using the riboFECT CP Transfection Kit according to the manufacturer's instructions.

MSC-exo extraction and identification
After transfection, the medium was collected and exosomes isolated using Exosome Isolation Reagent (Ruibo Biotechnology) according to the manufacturer's instructions.The collected exosomes were identified by Western blot detection of markers CD9, CD63, and CD81, and by analyzing shape and size by electron microscopy.Subsequently, the expression levels of miR129-5p and exo-con were confirmed by RT-PCR.

Animals
All animal experiments were approved by the Ethics Committee on Experimental Animals of Xuzhou Medical University (Approval number 202203A020).Adult C57/BL6J male mice (8 weeks of age) weighing 20-25 g were provided by the Experimental Animal Center of Xeuzhou Medical University, China, and housed in a specific pathogen-free (SPF) environment at 22 ± 2 °C under a 12-h/12-h light/dark cycle.

Kainic acid-induced SE and exosome injection
Male mice were randomly divided into four groups (n = 5/ group): CON, KA, KA + Exo-con, and KA + Exo-miR129-5p.Mice allocated to the 3 KA groups were administered 25 mg/kg KA (Sigma-Aldrich, USA) by intraperitoneal injection while CON group mice were injected with equal volume saline.Seizures were classified according to the Racine scale [27] as follows: grade 0, no response; grade 1, facial myoclonus; grade 2, head nodding; grade 3, forelimb clonus; grade 4, rearing and severe forelimb clonus; grade 5, rearing, falling, and severe forelimb clonus.Only mice exhibiting stage 3-5 seizures were considered eligible for continued treatment, other mice were excluded from experiments.After SE, mice exhibited a latent seizure-free phase.Diazepam (10 mg/kg, i.p.) was administered 1 h after KA to terminate convulsions.Mice of the KA + Exo-con and KA + Exo-miR129-5p groups were administered exo-con and exo-miR129-5p (100 μg in PBS), respectively, by tail vein injection 24 h after KA injection.

Western blotting
Hippocampal tissues were extracted from mice following the indicated treatment protocol (Sect.4.4), incubated in RIPA lysis buffer (Beyotime, Haimen, China) supplemented with the protease inhibitor PMSF, homogenized by homogenizer and centrifuged at 12000 rpm for 15 min at 4 °C.Subsequently, the supernatant was collected and the protein concentration determined using a BCA protein assay kit (Beyotime).Lysate samples were mixed with 5 × (v/v) loading buffer and boiled in a water bath for denaturation.The denatured protein samples (100 μg per gel lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes.Membranes were blocked with 5% non-fat milk, incubated with rabbit anti-HMGB1 monoclonal antibody (1:1000, abcam, UK) and rabbit anti-TLR4 polyclonal antibody (1:1000, abcam, UK) overnight at 4 ℃, rinsed with tris-buffered saline plus Tween-20 (TBST), incubated with Dylight 800 goat anti-rabbit IgG at room temperature (r/t) for 1.5 h, and rinsed again in TBST.Immunolabeling was recorded using an Odyssey scanner (LI-COR, USA) and quantified using ImageJ (NIH, Bethesda, MD, USA).

Real-time PCR
Total RNA was extracted from mouse hippocampus using the RNA Easy Fast Tissue/Cell Kit (Tiangen, Beijing, China) according to the manufacturer's instructions and reverse transcribed to cDNA using the PrimeScript RT reagent kit (Takara, Dalian, China) following the manufacturers' instructions.The total reaction system contained 2 μL cDNA, 0.4 μL forward primer, 0.4 μL reverse primer, and 10 μL SYBR Premix ExTaq II (Takara, Dalian, China).The reaction conditions for measuring miR129-59 expression were 35 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and termination at 95 °C for 15 s.The primers for RT-qPCR were as follows: F: 5′-ACC CAG TGC GAT TTG TCA -3′, R: 5′-ACT GTA CTG GAA GAT GGA CC-3′.

Enzyme-linked immunosorbent assays (ELISAs)
Hippocampal tissues were dissociated and collected 48 h after KA induction.The hippocampal concentrations of IL-1β, IL-6, and TNF-α were measured using ELISA kits (Jianglaibio, Shanghai, China) according to the manufacturer's protocol.In brief, hippocampal lysates were incubated with reaction buffer with five holes, followed by incubation for 2.5 h at r/t.Terminating the reaction after 30 min of substrate coloration.Absorbance was measured using a Synergy2 microplate reader (BIO-TEK) and converted to pg per mg of total protein.

Nissl and FJB staining
Mice were perfused through the heart with 4% polyformaldehyde 48 h after KA induction and brain tissues isolated, paraffin-embedded, and sectioned at 5 μm thickness.Sections were dehydrated, treated with Nissl staining solution at 37 °C for 10 min, immersed in 70% alcohol for 5 s, mounted with neutral balsam, and imaged using an IX71 microscope (Olympus, Tokyo, Japan).The number of Nissl-positive neurons in the hippocampal CA3 area was calculated using ImageJ software.

Image capture
All the immunostaining images were captured using an IX71 fluorescence microscope (Olympus, Tokyo, Japan) under different magnifications.The images of IBA-1, GFAP fluorescent staining, Nissl staining and FJB staining were captured at 20 × objective, while BrdU staining image were captured under 10 × objective.

Statistical analysis
Statistical Package for Social Sciences (SPSS) software version 20 was used for the statistical analysis.The datasets were examined for normality using a Q-Q plot test.Two groups were compared by Student's T-test and three or more groups by one-or two-way ANOVA followed by Tukey's post hoc tests.All data are presented as the mean ± SEM.A p < 0.05 was considered statistically significant for all tests.

Elevated HMGB1 expression and reduced miR129-5p expression in the hippocampus of SE model mice
Western blotting of hippocampal tissue extract from the four mouse groups 12, 24, 48, 72, and 96 h after KA injection revealed that SE significantly enhanced HMGB1 expression, with upregulation peaking at 48 h (Fig. 1A, B) conducted via ANOVA (F = 626.32,df = 24, p = 0.0001).In contrast, qPCR revealed that miR129-5p expression was downregulated by SE (Fig. 1C), with lowest expression at 48 h after KA injection, conducted via ANOVA (F = 373.61,df = 24, p = 0.0001).These findings suggest that miR129-5p serves to inhibit HMGB1 expression in the hippocampus and that preventing miR129-5p downregulation may suppress the expression of HMGB1, a potential mediator of SE-induced neuroinflammation and neuronal damage.Accordingly, in the subsequent experiment, we collected samples for detection at 48 h after SE.

Exo-miR129-5p injection decreased neural progenitor cell proliferation in the DG following SE
Finally, BrdU staining revealed that KA-induced SE markedly enhanced the rate of neural progenitor cell proliferation (Fig. 6A, B), and this response that was reduced by

Discussion
Neuroinflammation is a key pathophysiological characteristic of the epileptic brain, and in some cases, inflammation contributes to the progression and recurrence of epilepsy.Annamaria et al. proposed that targeting neuroinflammationrelated pathways may be an effective anti-epileptogenic and disease-modifying strategy [8].Indeed, anti-inflammatory drugs have demonstrated therapeutic efficacy on drug-resistant seizures [8].Moreover, anti-inflammatory interventions are reported to improve the disease course, reduce seizure frequency, and promote neuroprotection in animal models of epilepsy [28].Here we show that miRNA-mediated downregulation of the inflammatory signaling factor HMGB1 can suppress SE-induced hippocampal inflammation and neuronal death.
Consistent with our results, HMGB1 was reported to be upregulated in both animal models of epilepsy and epileptic patients [29,30], suggesting miRNA-mediated suppression of HMGB1 expression as a potential therapeutic strategy for SE-associated neuroinflammation and neurodegeneration.However, delivery of exogenous miRNAs is hampered by the instability of these molecules in the extracellular environment.Exosomes are important conduits for cell-to-cell communication by transporting proteins, miRNA, circRNA, lncRNA, and other components [26], and numerous studies support the therapeutic potential of exosomes loaded with various protective factors.Mesenchymal stromal cells can be conveniently isolated from various tissues and manipulated to release microsomes enriched in therapeutic factors.Moreover, MSCs naturally release a variety of neuroprotective factors [31].Accordingly, MSC-derived exosomes have garnered intense interest as sources for therapeutic exosomes.Here, we extracted exosomes from MSCs transfected with miR129-5p mimic and found that these exosomes were enriched in miR129-5p.Moreover, systemic injection of these exosomes increased miR129-5p expression in the hippocampus, supporting the feasibility of therapeutic application.Consistent with this notion, Li and colleagues reported that increased miR129-5p expression ameliorated neuroinflammation after ischemia-reperfusion by inhibiting HMGB1 and the TLR3-cytokine pathway [32].The current findings expand the spectrum of potential therapeutic applications to SE-associated neuroinflammation through downregulation of hippocampal HMGB1 and TLR4.
High mobility group box 1 protein is released from neurons, astrocytes, and microglia under pathological conditions and activates the TLR4 signaling pathway, triggering neuroinflammation.Status epilepticus results in activation of microglia and astrocytes and ensuing release of inflammatory factors [10,33,34].Immunostaining for markers of activated microglia and astrocytes revealed that the SEinduced increases in expression were reversed by exo-miR-129-5p concomitant with reductions in IL-1β, IL-6, and TNF-α concentrations.Collectively, these results strongly suggest that miR129-5p suppresses SE-induced neurodegeneration by inhibiting HMGB1/TLR4 and downstream neuroinflammatory pathways.
Chronic neuroinflammation may damage neurons by promoting hyperexcitability, initiating a perpetuating cycle of increased seizure activity, neuroinflammation, and neuronal damage [35,36].Neuroinflammation can in turn trigger hippocampal neurogenesis [10].Here, we report that systemic exo-miR129-5p administration reduced neuronal damage in CA3 region as evidenced by Nissl and FJB staining.Here, we observed no significant difference of neurons in CA1 region after SE induction, which may be due to differences in molding methods or drug dosage.In addition exo-miR129-5p reduced neurogenesis in the DG as evidenced by BrdU labeling.There is compelling evidence that proliferation of progenitor cell contributes to the changes in circuit structure underlying further seizure activity [11,37].Accordingly, this decrease in neurogenesis may prevent the progressive increase in seizure frequency and severity observed among many TLE patients.These findings provide evidence that miR129-5p-loaded exosomes may be a promising strategy to relieve pathological damage in epileptic brain.Additionally, the small sample size can be a limitation in our study, we will expand the sample size for further in-depth research.In this study, we mainly focus on neuroinflammation, and further exploration is needed to determine whether there is inhibitory effect on epileptic discharge and neuron activities.

Conclusion
In conclusion, we demonstrate miR129-5p expression is reduced in the hippocampus of SE model mice and that prevention of this decrease by systemic administration of MSC-derived miR129-5p-enriched exosomes can prevent SE-induced neurological damage by suppressing the proinflammatory HMGB1/TLR4 signaling axis.

Fig. 1
Fig. 1 Downregulation of miR129-5p expression and concomitant upregulation of HMGB1 protein in the mouse hippocampus following KA-induced SE.A Hippocampal HMGB1 expression was detected at 12, 24, 48, 72, and 96 h after KA injection by western blot.B Changes in HMGB1 expression over time as measured by relative

Fig. 2
Fig. 2 Extraction and identification of miR129-5p-loaded exosomes.A Detection of miR129-5p overexpression in transfected MSCs by qPCR.B Expression levels of exosomal protein markers CD9, CD63, and CD81 measured by western blotting.C Extracellular vesicle size determination by electron microscopy.The size distribution

Fig. 4
Fig. 4 Injection of exo-miR129-5p suppressed SE-induced hippocampal inflammation.A, B Immunofluorescence staining of the reactive microglial marker IBA-1 and reactive astrocyte marker GFAP in CA1 and CA3 regions of hippocampus.C, D Statistical histogram

Fig. 5 Fig. 6
Fig. 5 Injection of exo-miR129-5p prevented hippocampal neuronal damage following SE.A, B Representative images of Nissl staining and FJB staining in the hippocampal CA3 region.C, D Statistical his-